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TRANSCRIPTIONDEPARTMENT OF BIOCHEMISTRY

SUSAN B. CACERES-TENGCO, M.D.


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OBJECTIVES

DISCUSS the importance of the transcription

process

DIFFERENTIATE RNA molecules synthesized in the

mammalian cell from those made in prokaryotic cells

GIVE AND DESCRIBE the role of the requirements

for RNA synthesis :

- give the role of the template strand and codingstrand


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DESCRIBE the process/mechanisms including all thefactors and enzymes involved in the following steps ofRNA synthesis :

- initiation- elongation

- termination

DISCUSS the role of the different transcription signals :

TATA or Pribnow box Initiation and termination signals

Enhancers and silencers


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,

DEFINE introns and exons, cis and trans elements,

promoters, enhancers, transcription factors

DESCRIBE the processing of the primary transcript

(hnRNA) to mature mRNA in eukaryotes and give its

importance

DISCUSS how drugs can interfere with the

replication process


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IMPORTANCE OF TRANSCRIPTION

Errors and changes in synthesis, processing and

splicing of mRNA transcripts are causes of mutation

and diseases

TRANSCRIPTION :

Transfer of genetic information from DNA to RNA


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DEFINITION OF TERMS

CISELEMENT OR CIS-ACTING FACTOR

- short sequence of DNA important for regulating

the expression of a nearby gene

TRANS-ACTING FACTOR

- factors which are usually proteins that bind to the

cis element or some other DNA sequence, and in so

doing regulate gene expression


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PROMOTER

- short sequence of DNA to which RNA Polymerase

binds which ensures the right transcription start site

- are cis-acting elements

TATA or ATA BOX or PRIBNOW BOX

- short DNA sequence found in almost all promoters

for RNA Polymerase, often located -30 nucleotidesupstream of the start site in eukaryotes


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CAAT AND GC BOXES

- short sequences of DNA containing CAAT and GTC basesfound upstream from many promoters of RNA Pol II.Upstream location varies

TRANSCRIPTION FACTORS- proteins that bind to promoters and stimulate the activityof RNA Pol

- examples of trans-acting factors

ENHANCERS AND SILENCERS- DNA sequences which are not promoters but stimulate orinhibit promoter function. May act even if distant frompromoter


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CLASSES OF RNA

IMPORTANT FOR PROTEIN SYNTHESIS :

a. mRNA

b. tRNA

c. rRNA

IMPORTANT FOR SPLICING AND GENE

REGULATION :a. snRNA

b. micRNA


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SIMILARITIES BETWEEN RNA AND DNA

SYNTHESIS

Same steps : initiation, elongation and termination

Polarity is 5 to 3 Follows rules of base complementarity

Involves large, multicomponent initiation complexes


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DIFFERENT FROM DNA SYNTHESIS BY

THE FOLLOWING POINTS :

Uses ribonucleotides

U replaces T as base pair for A No primer involved

Only a portion of DNA is transcribed

No proofreading function


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TRANSCRIPTION UNIT

- DNA region that includes signals for initiation,elongation and termination

TRANSCRIBED STRAND OF DNA

- Template/sense strand (read in 3 to 5 direction)

ENZYME

- DNA directed (or dependent ) RNA Polymerase


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TEMPLATE STRAND


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BACTERIAL RNA POLYMERASE


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MAMMALIAN RNA POLYMERASES

RNAP I - synthesis of rRNA

RNAP II - synthesis of mRNA, most sensitive to

-amanitin toxin

RNAP III - synthesis of tRNA


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METHODS TO IDENTIFY

TRANSCRIPTION UNITS OF DNA

+1 - start of transcription unit ( corresponds

to 5 terminal of mRNA)

- region beyond this point isDOWNSTREAM

-1 - nucleotide adjacent to the transcription

initiation site which marks the promoter

region- negative numbers increase as it

proceeds UPSTREAM


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PROMOTERS

Marks where transcription is to start

In eukaryotes aside from conferring FIDELITY ( TATAbox ), also determines HOW FREQUENTLY

( GC/CAAT boxes or promoter proximal elements )

transcription is to occur


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BACTERIAL PROMOTERS


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BUT:

Eukaryotic RNAP cannot recognize promoter and

non-promoter sequences of DNA and has no sigmafactor to guide it to the proper start site

THUS:

proteins or trans-elements GENERAL

TRANSCRIPTION FACTORS (GTFs) facilitatepromoter-specific binding of RNAP


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CLASSES OF TRANSCRIPTION

FACTORS FOR CLASS II GENES


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EUKARYOTIC PROMOTERS (cis) AND

THEIR BINDING FACTORS (trans)


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TFIID - consists of TATA Binding Protein

(TBP) + 14 TBP-associated factors (TAFs)

- binds to the TATA box promoter

element

- is the only General Transcription

Factor (GTF) capable of binding to specific

sequences of DNA


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EUKARYOTIC BASAL TRANSCRIPTION

COMPLEX


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STEPWISE ASSEMBLY AND

RECRUITMENT HYPOTHESIS


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TRANSCRIPTION CONTROL REGIONS

IN EUKARYOTIC CELLS

Inr - -3 to +5, binds TFIID

DPE - +25, binds TFIID


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TRANSCRIPTION CONTROL REGIONS

TATA - fidelity of transcription

GC/CAAT - frequency

ENHANCERS OR REPRESSORS (SILENCERS) -

can increase or decrease rate of transcription

in response to various signals : heat shock, heavymetals, hormones


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REQUIREMENTS FOR TRANSCRIPTION

DNA- directed RNA Polymerase ( RNAP )

- enzyme of transcription

Sigma factor - helps RNAP to recognize and

bind to the promoter region (for prokaryotes)

Rho factor - termination protein (present

also in prokaryotes only)

General transcription factors ( GTF ) eukaryoticcells


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PROKARYOTIC TRANSCRIPTION


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TERMINATION SIGNALS OF

TRANSCRIPTION IN PROKARYOTES


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CLEAVAGE SIGNAL IN EUKARYOTES

No known rho factor in eukaryotes

AAUAAA consensus sequence may be the

termination signal

RNA endonuclease cleaves the transcript 15 bases

3 from this sequence


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TREX COMPLEX

Couples/links transcription, RNA splicing and

modification and nuclear export

Large fraction of nuclear RNA (50 75%) do not

contribute to cytoplasmic RNA


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ALTERNATIVE SPLICING MAKES FOR

DIFFERENT mRNAs (VARIABILITY)


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CONSENSUS SEQUENCE IN SPLICE

SITE


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SPLICING OF GLUCOKINASE IN LIVER

AND IN PANCREATIC CELLS


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DRUGS/CHEMICALS THAT AFFECT

TRANSCRIPTION

ACTINOMYCIN D - intercalates between two G-C

base pairs and effectively blocks chain elongation

RIFAMPICIN - binds to the subunit of bacterial

RNAP blocking formation of initiation complex

-AMANITIN - toxic poison of Amanita Phalloides

mushroom inhibits translocation of nuclear

eukaryotic RNAP II. No effect on bacterial,mitochondrial and plant polymerases


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